Hirschsprung disease (HSCR) is a leading cause of intestinal dysmotility resulting from a reduced enteric nervous system (ENS) and leading to lack of distal intestine innervations. HSCR can be caused by mutations in several different loci and shows considerable phenotypic variation: individuals carrying the same allele can differ in the extent of intestinal denervation and severity of enterocolitis, intestinal inflammation of unknown etiology that is a serious HSCR complication. The sources of these phenotypic variations in HSCR are not well understood. If we could identify factors that make some individuals healthier than others, we would gain insight into potential therapies for those with more serious disease. We have a panel of zebrafish ENS mutants that serve as models for understanding variation in HSCR. Zebrafish is an excellent model in which to study HSCR phenotypic variation because we can manipulate all of the relevant parameters. Our zebrafish mutants have different extents of distal intestine denervation, often with considerable phenotypic variation among mutants that share the same genotype. These mutants also exhibit variation in intestinal motility, variation in their intestinal bacterial communities, and variation in intestinal inflammation. We propose that alterations in the ENS phenotype in ENS mutants, including changes in specific neuronal subtypes, result in dysmotility that causes changes in the milieu of the intestinal lumen. This, in turn, can lead to formation of altered bacterial communities that can cause inflammation by recruiting neutrophils, cells of the innate immune system that respond to bacteria and are a hallmark of intestinal inflammation. We also propose that changes in the intestinal bacterial community composition and in inflammation can feed back to the ENS to amplify variation in intestinal motility. We will test these hypotheses by manipulating the ENS phenotype, intestinal bacterial composition, and neutrophil influx and comparing all of these processes in individual animals of known genotypes. We will follow bacterial colonization, neutrophil recruitment, and intestinal motility in living animals in real time. Our proposed studies will provide new information about how the ENS regulates the composition of the intestinal bacterial community and inflammation, and how these processes feed back onto the ENS to amplify intestinal pathology. This information will provide new insights into the mechanisms of phenotypic variation in HSCR, expand our understanding of phenotypic variation in human disease, and may reveal new targets for therapies.